Cellulase preparation containing nonionic surfactant and method of treating fiber

ABSTRACT

The present invention relates to a cellulase preparation comprising nonionic surfactants together with endoglucanases derived from Zygomycetes; a detergent composition comprising the above cellulase preparation with detergent components; a method of treating fabric which comprises treating cellulose-containing fabric with the above cellulase preparation so as to improve the properties of the fabric; a method of deinking waster paper which comprises a step of treating waste paper with the above cellulase preparation together with a deinking agent; and a method of improving the freeness of paper pulp which comprises a step of treating paper pulp with the above cellulase preparation.

TECHNICAL FIELD

[0001] The present invention relates to a cellulase preparation comprising a nonionic surfactant(s) by which endoglucanase activity has improved; a detergent comprising the cellulase preparation; and a method of treating fabric, which uses the cellulase preparation.

BACKGROUND ART

[0002] Cellulase has three types of enzyme activities: cellobiohydrolase activity which hydrolyzes the solid crystal regions of cellulose from the nonreduced end in an exo manner so as to generate cellobiose; endoglucanase activity which hydrolyzes the amorphous regions of cellulose in an endo manner so as to make cellulose molecules into low molecular weight molecules and to generate various types of cellooligosaccharides; and β-glucosidase activity which decomposes cellobiose or cellooligosaccharide into glucose. Of the enzymes, when endoglucanase exerts high activity, cellulase is advantageously used to treat fabric.

[0003] To impart certain desired properties to cellulose-containing fabric, the fabric has conventionally been treated with cellulase. For example, in the textile industry, treatment with cellulase is carried out to improve the touch and appearance of cellulose-containing fabric, or to impart “stonewash” appearance to colored cellulose-containing fabric thereby providing the fabric with localized color variation (EP Patent No. 307,564).

[0004] It is known that fuzz is developed on colored cellulose-containing fabric by repeated washing and that it blurs the color of the colored fabric. A cellulase-containing detergent removes such fuzz and makes the color of the fabric clear (color clarification) (EP Patent No. 220,016), and therefore detergents containing cellulase are currently on the market mainly in Europe and the United States.

[0005] In such textile processing, cellulase derived from wood-rotting fungi such as Trichoderma and Humicola is mainly used. Such cellulase is used as a mixture comprising multiple cellulase components which is obtained by processing a culture filtrate of microorganisms having cellulolytic activity. However, in order to achieve greater economy, a cellulase preparation obtained by isolating from cellulase components, only endoglucanase which largely acts on fabric treatment, and genetically enhancing it, has recently been used. Examples of such endoglucanase with high activity include: EGV (JP Patent Publication (PCT Translation) No. 5-509223) and NCE4 (WO98/03640) derived from Humicola insolens, which strongly act on cotton fabrics; RCE I, RCE II and RCE III derived from Rhizopus oryzae, which strongly act on lyocell fabrics; MCE I and MCE II derived from Mucor circinelloides; and PCE I derived from Phycomyces nitens (WO00/24879).

[0006] In order to improve the effects of cellulase, the combined use of additives has also been attempted. For example, JP Patent Publication (PCT Translation) No. 5-507615 describes that a water-soluble polymer such as polyvinylpyrrolidone, polyvinyl alcohol and polyacrylamide enhances the effects of Humicola insolens-derived cellulase and improves its activity of removing fuzz from colored fabric. Moreover, it is known that CMCase activity in the culture solution of Trichoderma viride is improved by the addition of Tween 20 (Ooshima, H. et al., Biotechnology and Bioengineering 28: 1727-1734, 1986).

[0007] However, the cellulases used for the above-described purposes are all expensive. Therefore, in order to achieve an industrial level application, further improvement of endoglucanase activity is desired, so that the above effects of cellulase can be more efficiently exerted.

DISCLOSURE OF THE INVENTION

[0008] It is therefore the object of the present invention to provide a cellulase preparation having improved endoglucanase activity, which can be used for the purpose that the fabric treatment for improving cellulose-containing fabric such as the removal of fuzz can be carried out efficiently and economically.

[0009] As a result of intensive studies directed towards the above object, the present inventors have found that a nonionic surfactant enhances the effects of Zygomycetes-derived endoglucanases such as RCE I, MCE I and PCE I, at rates far higher than Trichoderma- or Humicola-derived endoglucanase, thereby completing the present invention.

[0010] That is to say, the present invention relates to the following (1) to (5):

[0011] (1) A cellulase preparation comprising nonionic surfactant(s) together with endoglucanase(s) derived from Zygomycetes,

[0012] (2) A detergent composition obtained by blending said cellulase preparation with detergent components,

[0013] (3) A method of treating fabric which comprises treating cellulose-containing fabric with said cellulase preparation so as to improve the properties of the fabric,

[0014] (4) A method of deinking waste paper comprising a step of treating waste paper with said cellulase preparation together with a deinking agent, and

[0015] (5) A method of improving the freeness of paper pulp comprising a step of treating paper pulp with said cellulase preparation.

[0016] [1] Cellulase Preparation

[0017] The cellulase preparation of the present invention comprises endoglucanase(s) derived from Zygomycetes, and nonionic surfactant(s).

[0018] In the present invention, the term, endoglucanase, is used to mean endo-1,4-β-glucanase EC3.2.1.4, and it has the activity to hydrolyze β-1,4glucopyranosil bonds of β-1,4-glucan.

[0019] Examples of endoglucanases derived from Zygomycetes used in the present invention include endoglucanases derived from Rhizopus sp., Phycomyces sp. or Mucor sp. Specific examples of such endoglucanases used herein include RCE I, RCE II, RCE III, MCE I, MCE II and PCE I proteins having amino acid sequences as shown in SEQ ID NOS: 1 to 6, respectively, which are disclosed in WO00/24879.

[0020] RCE I, II and III are derived from the Rhizopus oryzae CP96001 strain, which has been deposited under the terms of the Budapest Treaty with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), [the former name: the National Institute of Bioscience and Human-Technology, Agency of. Industrial Science and Technology, the former address: Higashi 1-1-3, Tsukuba, Ibaraki, Japan] under accession No. FERM BP-6889 on Apr. 21, 1997. MCE I and II are derived from the Mucor circinelloides CP99001 strain, which has been deposited with the same international depositary authority, that is, the National Institute of Advanced Industrial Science and Technology, an Independent. Administrative Institution under the Ministry of Economy, Trade and Industry, at the AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), [the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, the former address: Higashi 1-1-3, Tsukuba, Ibaraki, Japan] under accession No. FERM BP-6890 on July 2, 1999. Moreover, PCE I was derived from the Phycomyces nitens CP99002 strain, which has been deposited with the same international depositary authority under accession No. FERM BP-6891 on Jul. 2, 1999.

[0021] In the present invention, the above-stated RCE I, RCE II, RCE III MCE I, MCE II and PCE I proteins include modified proteins and homologues thereof. The term “modified protein” is used herein to mean a protein having an amino acid sequence comprising an addition, insertion, deletion or substitution of one or more amino acids (for example, one to several tens of, specifically, one to approximately fifty, preferably, one to approximately thirty, and more preferably, one to approximately nine amino acids) with respect to the amino acid sequence of each of the above mentioned RCE I, RCE II, RCE III, MCE I, MCE II and PCE I, and having endoglucanase activity. The term “homologue” is used herein to mean a protein having an amino acid sequence encoded by a gene (nucleotide sequence) complementary to a gene (nucleotide sequence) “hybridizing under stringent conditions” with a gene (nucleotide sequence) encoding the amino acid sequence of each of the above mentioned RCE I, RCE II, RCE III, MCE I, MCE II and PCE I, and having endoglucanase activity. Herein, the term “under stringent conditions” is used to mean conditions in which a probe comprising a nucleotide sequence encoding a part or all of the amino acid sequences of RCE I, RCE II, RCE III, MCE I, MCE II or PCE I, or the amino acid sequences of modified proteins thereof would hybridize with a gene encoding a homologue, but the same probe does not hybridize with an endoglucanase NCE 4 gene (SEQ ID NO: 7) as disclosed in WO98/03640 or endoglucanase SCE 3 gene (SEQ ID NO: 8) as disclosed in WO98/54322 (it should be noted that the amount of DNA used herein is equivalent to the amount of each of the NCE 4 gene, the SCE 3 gene and a gene encoding the homologue.) More specifically, it means conditions in which, using as a probe a full-length DNA sequence encoding the amino acid sequence of labeled RCE I, pre-hybridization is carried out at 42° C. for 1 hour according to the method of the ECL direct DNA/RNA labeling and detection system (Amersham), then the above probe is added thereto followed by hybridization at 42° C. for 15 hours, and thereafter, the resultant product is washed twice with a solution containing 0.4% SDS, 6M urea and 0.5×SSC (SSC; 15 mM trisodium citrate, 150 mM sodium chloride) at 42° C. for 20 minutes, and finally followed by washing the product twice with 5× SSC at room temperature for 10 minutes.

[0022] An example of such a modified protein or homologue includes a protein comprising an amino acid sequence having preferably 80% or more homology, more preferably 90% or more homology, further more preferably 95% or more homology, and most preferably 98% or more homology with the amino acid sequence of RCE I, RCE II, RCE III, MCE I, MCE II or PCE I. The above-stated values of homology may be calculated by a homology search program which is well known to a person skilled in the art, but the values are preferably calculated using a default (initialized) parameter of FASTA3 [Science, 227, 1435-1441 (1985); Proc. Natl. Acad. Sci. USA, 85, 2444-2448 (1998);

[0023] http ://www.ddbj.nig.ac.jp/E-mail/homology-j.html].

[0024] The nonionic surfactant included in the cellulase preparation of the present invention means a surfactant whose hydrophilic group is a nonionic group. Examples of such a nonionic surfactant include polyoxyethylene alkylether, polyoxyethylene alkylphenylether, polyoxyethylene monofatty acid ester, polyoxyethylene sorbitan monofatty acid ester, sorbitan monofatty acid ester, polyethylene glycol, glycerol monofatty acid ester, polyglycerine fatty acid ester, alkyl glycoside, esters of polyethoxylated alkyl glycosides, alkyl dimethyl amine oxide, fatty acid diethanol amide, polyoxyethylene alkylamine, terephthalic acid tetraethylene glycol polymer, alkyl polyethylene glycol ether, nonyl phenol polyethylene glycol ether, and fatty acid ester of sucrose or glucose. These nonionic surfactants can be used alone or in combination with other nonionic surfactants.

[0025] Moreover, the cellulase preparation of the present invention may comprise components which are conventionally contained in cellulase preparations such as excipients and preservatives. The form of the cellulase preparation may be solid or liquid, and specific examples of the form include powder, particulate, granule, non-dusting granule and liquid formulation.

[0026] A non-dusting granule that is one form of cellulase preparation can be produced according to the common dry granulation method. That is to say, powder cellulase enzyme is mixed with one or several types selected from a group comprising inorganic salts such as sodium sulfate and sodium chloride which are neutral and do not have an effect on endoglucanase activity; minerals such as bentonite and montmorillonite which do not have an effect on endoglucanase activity; and neutral organic matters such as starch and powder cellulose. Thereafter, the powders or the finely suspended suspension of one or several types of the above-described nonionic surfactants which improve the effects of endoglucanase are added to the mixture, and the thus obtained product is then fully mixed or kneaded. Depending on the situation, a synthetic polymer such as polyethylene glycol and natural polymer such as starch, which binds solids, is optionally added to the mixture and further kneaded. Thereafter, granulation is carried out by extrusion molding, using, for example, a disk pelleter, and the obtained molded material is then converted into a spherical form using a marumerizer followed by drying, so that non-dusting granules can be produced. Naturally, it is also possible to coat the surface of granules with a polymer or the like to control the permeation of oxygen or water. This time, one or multiple nonionic surfactants which improve the effect of endoglucanase are added to the above cellulase preparation at a ratio of 0.1 to 50% by weight, preferably 0.1 to 30% by weight, and more preferably 1 to 20% by weight.

[0027] On the other hand, the liquid preparation can be prepared by blending an endoglucanase stabilizer such as a synthetic and natural polymer with a cellulase solution, and adding inorganic salts or a synthetic preservative, as necessary. This time, one or multiple nonionic surfactants which improve the effect of endoglucanase can also be added. As in the case of the non-dusting granule, one or multiple nonionic surfactants which improve the effect of endoglucanase are added to the above cellulase preparation at a ratio of 0.01 to 50% by weight, preferably 0.1 to 30% by weight, and more preferably 1 to 20% by weight.

[0028] [2] Detergent Composition

[0029] The above described cellulase preparation of the present invention is blended with known detergent components such as builders, bleaching agents, bleaching activators, corrosion inhibitors, sequestering agents, stain dissociating polymers, aromatics, other enzymes, enzyme stabilizers, formulation assistants, fluorescent brightening agents and foaming promoters so that a detergent composition can be produced.

[0030] The present detergent composition relates to the granular soil removal, the color clarification, the defuzzing, the depilling and the reduction of stiffness, and these can be improved by the composition of the present invention.

[0031] [3] Method of Treating Fabric

[0032] The method of treating fabric of the present invention comprises treating cellulose-containing fabric with the above cellulase preparation.

[0033] The following properties of cellulose-containing fabric can be improved by the present fabric treatment method:

[0034] (1) Removal of fuzz (reduction of the rate of the formation of fuzz, and reduction of fuzz),

[0035] (2) Color clarification of colored cellulose-containing fabric,

[0036] (3) Providing of localized color variation to colored cellulose-containing fabric, that is, providing of stonewash-like appearance and texture to colored cellulose-containing fabric, typically jeans,

[0037] (4) Enhancement of the touch and appearance of fabric by reducing weight, and

[0038] (5) Softening of fabric (reduction of stiffness).

[0039] The above method of treating fabric can be carried out typically during washing, but it can also be carried out during soaking or rinsing. Specifically, the method of treating fabric of the present invention can be carried out by adding the cellulase preparation of the present invention into water in which fabric is or will be soaked.

[0040] Conditions such as contact temperature and the amount of endoglucanase may appropriately be determined, taking into consideration various other conditions. For example, in the case of reducing the rate of the formation of fuzz or reducing fuzz of the cellulose-containing fabric, the fabric can be treated at a temperature of approximately 30° C. to 60° C., using 10 to 10,000 mg/L of nonionic surfactants and endoglucanases in a protein concentration of 0.05 to 20 mg/L.

[0041] In the case of providing the colored cellulose-containing fabric with localized color variations, the fabric can be treated at a temperature of approximately 30° C. to 60° C., using 10 to 10,000 mg/L of nonionic surfactants and endoglucanases in a protein concentration of 0.1 to 30 mg/L.

[0042] In a processing of reducing weight which is directed towards the improvement of the touch and appearance of the cellulose-containing fabric, the fabric can be treated at a temperature of approximately 30° C. to 60° C., using 10 to 10,000 mg/L of nonionic surfactants and endoglucanases in a protein concentration of 0.2 to 50 mg/L.

[0043] In any of the above cases, the nonionic surfactant may be dissolved or suspended in water.

[0044] The protein concentration of each type of endoglucanase is measured by HPLC analysis using TSKgel TMS-250 column (4.6 mm I.D.×75 cm) (TOSOH Corporation). The HPLC analysis involves loading acetonitrile in 0.05% TFA (trifluoroacetic acid) with a linear concentration gradient of 0% to 80% at a flow rate of 1.0 ml/min so as to elute each type of endoglucanase, and calculating the protein concentration from the peak area at UV 280 nm. A purified NCE4, the protein concentration of which is previously determined by a Protein Assay Kit (BioRad Laboratories), is subjected to the HPLC analysis in the same manner as above, so that it is used as a standard. According to the method described in International Publication No. WO98/03640, the purified NCE4 is obtained by culturing Humicola insolens [Humicola insolens MN200-1 which was deposited under accession No. FERM BP-5977 (original accession No. FERM-15736, original accession date: Jul. 15, 1996) with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology], and purifying the obtained culture. As a standard for the determination of a protein concentration in the Protein Assay Kit, Albumin Standard (Bovin serum albumin, fraction V, PIERCE) is used.

[0045] [4] Method of Deinking Waste paper

[0046] The method of deinking waste paper of the present invention comprises treating waste paper with the above-described cellulase preparation together with a deinking agent.

[0047] Specifically, the present method can be carried out by treating waste paper with the cellulase preparation of the present invention together with a deinking agent in a deinking step in a process of producing recycled paper from waste paper. The present method enables the deinking of waste paper, thereby improving the whiteness of waste paper. Waste paper which is the target of the present method include all types of common waste paper such as used news paper, used magazine paper and low to middle grade of printed used paper comprising mechanical pulp and/or chemical pulp; used wood-free paper comprising chemical pulp; and printed waste paper thereof such as coating paper. The above-described deinking agent means an agent used in the deinking of waste paper, and examples of such a deinking agent include alkali such as sodium chloride and sodium carbonate, sodium silicate, hydrogen peroxide, phosphate, anionic or nonionic surfactant, scavenger such as oleic acid, assistant agents such as pH stabilizer, chelating agent and dispersing agent, and others.

[0048] [5] Method of Improving Freeness of Paper Pulp

[0049] The method of improving the freeness of paper pulp of the present invention comprises treating paper pulp with the above-described cellulase preparation.

[0050] Specifically, the present method can be carried out by treating paper pulp with the cellulase preparation of the present invention. Examples of paper pulp which can be the target of the present method include waste paper pulp, recycled paperboard pulp, kraft pulp, sulfite pulp, thermo-mechanical treatment pulp, and other high-yield pulp.

[0051] This specification includes the contents as disclosed in the specification of Japanese Patent Application No. 2000-343921, which is a priority document of the present application.

BEST MODE FOR CARRYING OUT THE PRESENT INVENTION

[0052] The present invention is further described in the following examples. However, the examples are provided for illustrative purposes only, and are not intended to limit the scope of the invention.

[0053] Hereinafter, the term “endoglucanase activity” means CMCase activity. Moreover, regarding the “CMCase activity,” when a solution comprising cellulase enzyme and carboxymethylcellulose (CMC, Tokyo Kasei Kogyo Co., Ltd.) is incubated for a certain time period and the amount of reducing sugar released is measured, the amount of enzyme producing the reducing sugar corresponding to 1 lmol of glucose per minute, is defined as 1 unit.

EXAMPLE 1

[0054] (Comparison Among Improvement Ratios of Fuzz-Removing Activities of Various Types of Cellulases by Addition of Nonionic Surfactant)

[0055] The cultivation of Rhizopus oryzae, Mucor circinelloides and Phycomyces nitens, and the purification of RCE I, MCE I and PCE I endoglucanases from the cultures, were carried out by the method described in International Publication No. WO00/24879.

[0056] The cultivation of Humicola insolens [Humicola insolens MN200-1 which was deposited under accession No. FERM BP-5977 (original accession No. FERM P-15736, original accession date: Jul. 15, 1996) with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology], and the purification of NCE4 endoglucanase from the culture, were carried out by the method described in International Publication No. WO98/03640.

[0057] The cultivation of Trichoderma viride [Trichoderma viride MC300-1 which was deposited under accession No. FERM BP-6047 (original accession No. FERM P-15842, original accession date: Sep. 9, 1996) with the National Institute of Advanced Industrial Science and Technology, at the an Independent Administrative Institution under the Ministry of Economy, Trade and Industry, AIST Tsukuba Central 6, Higashi 1-1-1, Tsukuba, Ibaraki, Japan (postal code No. 305-8566), the former name: the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology] was carried out by the method described in International Publication No. WO98/54332.

[0058] Fuzz-removing treatment from a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. was dyed in brown by reactive dyeing in Tsuyatomo-Senko), was carried out using the obtained culture supernatant and uniformly purified various endoglucanases under the following conditions.

[0059] (Test Conditions)

[0060] Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MFG., Japan)

[0061] Temperature: 40° C.

[0062] Time: 120 minutes

[0063] Amount of reaction solution: 40 ml

[0064] Reaction pH: only the Trichoderma viride culture supernatant was adjusted at pH 4 (20 mM acetate buffer), all other enzyme solutions were adjusted at pH 6 (5 mM phosphate buffer) for reaction. All the buffers were prepared using deionized water.

[0065] Type and amount of nonionic surfactant: 100 μg/ml polyoxyethylene lauryl ether (NOF Corporation, product name: NissanNonion K-220, number of oxyethylene units added: 20, HLB: 16.2)

[0066] To the treating solution were added four of about 16 g rubber balls together with the enzyme solution.

[0067] The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in each of both cases of adding and not adding the nonionic surfactant. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz in the case of not adding the nonionic surfactant by the amount in the case of adding the nonionic surfactant, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of the nonionic surfactant. The results are shown in Table 1. TABLE 1 Improvement ratio of fuzz-removing activity by addition of nonionic surfactant (fold) Humicola insolens culture 1.2 supernatant Trichoderma viride culture 1.33 supernatant Purified NCE 4 1.5 Purified RCE I 4.0 Purified MCE I 6.0 Purified PCE I 4.0

[0068] From the results of Table 1, it is found that the fuzz-removing activity of RCE I, MCE I and PCE I, which are endoglucanases derived from Zygomycetes, is improved by the addition of the nonionic surfactant at a level far higher than other enzymes including cellulase derived from Humicola insolens or Trichoderma viride, which are previous findings.

EXAMPLE 2

[0069] (Improvement Effect of Fuzz-Removing Activity of RCE I Expressed in Humicola by Addition of Various Nonionic Surfactants)

[0070] RCE I endoglucanase was expressed in Humicola insolens according to the method described in Examples D3 and 4 of International Publication No. WO00/24879. Fuzz-removing treatment from a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. was dyed in brown by reactive dyeing in Tsuyatomo-Senko), was carried out using the obtained culture supernatant under the following conditions.

[0071] (Test Conditions)

[0072] Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MFG., Japan) Temperature: 40° C.

[0073] Time: 120 minutes

[0074] Amount of reaction solution: 40 ml

[0075] Reaction pH: pH 6 (5 mM phosphate buffer prepared with deionized water)

[0076] Amount of nonionic surfactants: 100 μg/ml

[0077] Type of nonionic surfactants: Refer to Table 2 as described below.

[0078] To the treating solution were added four of about 16 g rubber balls together with the enzyme solution. TABLE 2 Type of surfactants Product names Manufacturing companies Polyoxyethylene lauryl Brij-35 Wako Pure Chemical ether (number of oxyethy- Industries, Ltd. lene units added: 4) Polyoxyethylene lauryl K-220 NOF Corporation ether (number of oxyethy- lene units added: 20) Polyoxyethylene cetyl ether P-208 NOF Corporation (number of oxyethylene units added: 8) Polyoxyethylene nonyl Nonipol 100 Sanyo Chemical phenyl ether Industries, Ltd. Polyoxyethylene octyl Triton X100 Wako Pure Chemical phenyl ether Industries, Ltd. Polyoxyethylene sorbitan Tween 80 Wako Pure Chemical monooleate Industries, Ltd. Polyethylene glycol 4000 PEG 4000 Wako Pure Chemical Industries, Ltd. Terephthalic acid tetra- FR 550 Goo Chemical Co., Ltd. ethylene glycol polymer

[0079] The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in each of both cases of adding and not adding each nonionic surfactant. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz in the case of not adding the nonionic surfactant by the amount in the case of adding the nonionic surfactant, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of the nonionic surfactant. The results are shown in Table 3. TABLE 3 Improvement ratio of fuzz-removing activity by addition of Type of nonionic surfactants nonionic surfactants (fold) Polyoxyethylene lauryl ether 3.0 (number of oxyethylene units added: 4) Polyoxyethylene lauryl ether 3.0 (number of oxyethylene units added: 20) Polyoxyethylene cetyl ether 2.0 (number of oxyethylene units added: 8) Polyoxyethylene nonyl 3.0 phenyl ether Polyoxyethylene octyl 2.0 phenyl ether Polyoxyethylene sorbitan 2.0 monooleate Polyethylene glycol 4000 2.0 Terephthalic acid tetraethylene 2.5 glycol polymer

[0080] From the results of Table 3, it is found that the fuzz-removing activity of the culture supernatant obtained by expressing and secreting RCE I in Humicola insolens was improved by any of the above nonionic surfactants.

EXAMPLE 3

[0081] (Improvement Effect of Fuzz-Removing Activity of RCE I Expressed in Humicola by Addition of Nonionic Surfactants with Various Concentrations)

[0082] RCE I endoglucanase was expressed in Humicola insolens according to the method described in Examples D3 and 4 of International Publication No. WO00/24879. Fuzz-removing treatment from a cotton knit fabric with fuzz formed in a large washer (a fabric of 6 cm×8 cm from Cotton Smooth Knit No. 3900, Nitto Boseki Co., Ltd. was dyed in brown by reactive dyeing in Tsuyatomo-Senko), was carried out using the obtained culture supernatant under the following conditions.

[0083] (Test Conditions)

[0084] Testing machine: Launder Meter L-20 (Daiei Kagaku Seiki MPG., Japan)

[0085] Temperature: 40° C.

[0086] Time: 120 minutes

[0087] Amount of reaction solution: 40 ml

[0088] Reaction pH: pH 6 (5 mM phosphate buffer prepared with deionized water)

[0089] Amount of nonionic surfactant: 10 to 10,000 μg/ml

[0090] Type of nonionic surfactant: polyoxyethylene lauryl ether (NOF Corporation, product name: NissanNonion K-220, number of oxyethylene units added: 20, HLB: 16.2)

[0091] To the treating solution were added four of about 16 g rubber balls together with the enzyme solution.

[0092] The amount of the enzyme solution required to remove approximately 50% of the formed fuzz on the basis of visual evaluation was determined in the case of adding the nonionic surfactant in each concentration. Thereafter, a value was obtained by dividing the amount of the enzyme solution required to remove approximately 50% of the fuzz in the case of not adding the nonionic surfactant, by the amount in the case of adding the nonionic surfactant in each concentration, and the obtained value was defined as an improvement ratio of the fuzz-removing activity by the addition of the nonionic surfactant in each concentration. The results are shown in Table 4. TABLE 4 Improvement ratio of fuzz-removing Additive amount of nonionic activity by addition of surfactant (μg/ml) nonionic surfactant (fold) 10 1.5 20 2.0 50 2.5 100 3.0 200 3.0 400 3.0 1000 3.0 2000 2.0 3000 1.5 5000 1.5 10000 1.5

[0093] From the results of Table 4, it is found that the fuzz-removing activity of the culture supernatant obtained by expressing and secreting RCE I in Humicola insolens was improved by the addition of the nonionic surfactant having a wide range of concentration from 10 to 10,000 μg/ml.

EXAMPLE 4

[0094] (Production of RCE I Cellulase Preparation Comprising Nonionic Surfactant)

[0095] After mixing the following raw materials, an appropriate amount of water was added thereto, and the mixture was kneaded. The obtained product was subjectetd to a disk pelleter for molding, and the product obtained by injection molding was converted in a particle form using a marumerizer (Fuji Paudal Co., Ltd.) followed by drying and sieving the product so as to obtain a granulated product. Mixing ratio (Raw materials) (%) S-220 (nonionic surfactant manufactured by NOF Corporation)   10% RCE I cellulase powder product   10% Magnesium chloride (Wako Pure Chemical Industries, Co., Ltd.)  0.5% Monopotassium phosphate (same as above)   2% Dipotassium phosphate (same as above)   1% Corn starch (Shikishima Starch Co.) 76.5%

[0096] The RCE I cellulase powder product was prepared by concentrating the culture supernatant of RCE I expressed in Humicola insolens using ultrafiltration, according to the method described in Examples D3 and 4 of International Publication No. WO00/24879, followed by spray drying.

[0097] All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

[0098] Industrial Applicability

[0099] The present invention provides a cellulase preparation having dramatically improved Zygomycetes-derived endoglucanase activity by adding a nonionic surfactant into the preparation. If the present cellulase preparation is used in the treatment of fabric such as the reduction of fuzz of cellulose-containing fabric, the improvement of touch and appearance, the color clarification, localized color variation or softening, the deinking of waste paper, or the processing of improving the freeness of paper pulp, each of the above treatments can be carried out with a less amount of enzyme, thereby significantly reducing cost.

1 8 1 338 PRT Rhizopus oryzae CP96001 sig_peptide (-23)...(-1) mat_peptide (1)...(315) 1 Met Lys Phe Ile Thr Ile Ala Ser Ser Ala Leu Leu Ala Leu Ala Leu -20 -15 -10 Gly Thr Glu Met Ala Ser Ala Ala Glu Cys Ser Lys Leu Tyr Gly Gln -5 1 5 Cys Gly Gly Lys Asn Trp Asn Gly Pro Thr Cys Cys Glu Ser Gly Ser 10 15 20 25 Thr Cys Lys Val Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Ser Gly 30 35 40 Ser Ser Gly Asn Lys Ser Ser Glu Ser Ala His Lys Lys Thr Thr Thr 45 50 55 Ala Ala His Lys Lys Thr Thr Thr Ala Ala His Lys Lys Thr Thr Thr 60 65 70 Ala Pro Ala Lys Lys Thr Thr Thr Val Ala Lys Ala Ser Thr Pro Ser 75 80 85 Asn Ser Ser Ser Ser Ser Ser Gly Lys Tyr Ser Ala Val Ser Gly Gly 90 95 100 105 Ala Ser Gly Asn Gly Val Thr Thr Arg Tyr Trp Asp Cys Cys Lys Ala 110 115 120 Ser Cys Ser Trp Pro Gly Lys Ala Asn Val Ser Ser Pro Val Lys Ser 125 130 135 Cys Asn Lys Asp Gly Val Thr Ala Leu Ser Asp Ser Asn Ala Gln Ser 140 145 150 Gly Cys Asn Gly Gly Asn Ser Tyr Met Cys Asn Asp Asn Gln Pro Trp 155 160 165 Ala Val Asn Asp Asn Leu Ala Tyr Gly Phe Ala Ala Ala Ala Ile Ser 170 175 180 185 Gly Gly Gly Glu Ser Arg Trp Cys Cys Ser Cys Phe Glu Leu Thr Phe 190 195 200 Thr Ser Thr Ser Val Ala Gly Lys Lys Met Val Val Gln Val Thr Asn 205 210 215 Thr Gly Gly Asp Leu Gly Ser Ser Thr Gly Ala His Phe Asp Leu Gln 220 225 230 Met Pro Gly Gly Gly Val Gly Ile Phe Asn Gly Cys Ser Ser Gln Trp 235 240 245 Gly Ala Pro Asn Asp Gly Trp Gly Ser Arg Tyr Gly Gly Ile Ser Ser 250 255 260 265 Ala Ser Asp Cys Ser Ser Leu Pro Ser Ala Leu Gln Ala Gly Cys Lys 270 275 280 Trp Arg Phe Asn Trp Phe Lys Asn Ala Asp Asn Pro Ser Met Thr Tyr 285 290 295 Lys Glu Val Thr Cys Pro Lys Glu Ile Thr Ala Lys Thr Gly Cys Ser 300 305 310 Arg Lys 315 2 366 PRT Rhizopus oryzae CP96001 sig_peptide (-23)...(-1) mat_peptide (1)...(343) 2 Met Lys Phe Ile Thr Ile Thr Ser Ser Ala Leu Leu Ala Leu Ala Leu -20 -15 -10 Gly Thr Glu Met Ala Ser Ala Ala Lys Cys Ser Lys Leu Tyr Gly Gln -5 1 5 Cys Gly Gly Lys Asp Trp Asn Gly Pro Thr Cys Cys Glu Ser Gly Ser 10 15 20 25 Thr Cys Lys Val Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Ala Pro Glu 30 35 40 Ser Asn Gly Asn Lys Ser Ser Glu Cys Ser Lys Leu Tyr Gly Gln Cys 45 50 55 Gly Gly Lys Asp Trp Asn Gly Pro Thr Cys Cys Glu Ser Gly Ser Thr 60 65 70 Cys Lys Val Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Ala Pro Glu Ser 75 80 85 Asn Gly Asn Lys Thr Ser Glu Ser Ala His Lys Thr Thr Thr Thr Thr 90 95 100 105 Ala Pro Ala Lys Glu Ile Thr Thr Thr Ala Lys Ala Ser Asn Ser Ser 110 115 120 Asn Ser Ser Gly Lys Tyr Ser Ile Val Ser Gly Gly Ala Ser Gly Asn 125 130 135 Gly Val Thr Thr Arg Tyr Trp Asp Cys Cys Lys Ala Ser Cys Ser Trp 140 145 150 Pro Gly Lys Ala Asn Val Ser Ser Pro Val Lys Ser Cys Asn Lys Asp 155 160 165 Gly Val Thr Ala Leu Ser Asp Ser Asn Val Gln Ser Gly Cys Asn Gly 170 175 180 185 Gly Asn Ser Tyr Met Cys Asn Asp Asn Gln Pro Trp Ala Val Asn Asp 190 195 200 Asn Leu Ala Tyr Gly Phe Ala Ala Ala Ala Ile Ser Gly Gly Gly Glu 205 210 215 Ser Arg Trp Cys Cys Ser Cys Phe Glu Leu Thr Phe Thr Ser Thr Ser 220 225 230 Val Ala Gly Lys Lys Met Val Ile Gln Val Thr Asn Thr Gly Gly Asp 235 240 245 Leu Gly Ser Ser Thr Gly Ala His Phe Asp Leu Gln Met Pro Gly Gly 250 255 260 265 Gly Val Gly Ile Phe Asn Gly Cys Ser Lys Gln Trp Gly Ala Pro Asn 270 275 280 Asp Gly Trp Gly Ser Arg Tyr Gly Gly Ile Ser Ser Ala Ser Asp Cys 285 290 295 Ser Ser Leu Pro Ser Ala Leu Gln Ala Gly Cys Lys Trp Arg Phe Asn 300 305 310 Trp Phe Lys Asn Ala Asp Asn Pro Ser Met Thr Tyr Lys Glu Val Thr 315 320 325 Cys Pro Lys Glu Ile Thr Ala Lys Thr Gly Cys Ser Arg Lys 330 335 340 3 360 PRT Rhizopus oryzae CP96001 sig_peptide (-23)...(-1) mat_peptide (1)...(337) 3 Met Lys Phe Leu Thr Ile Ala Ser Ser Ala Ile Leu Ala Leu Ala Val -20 -15 -10 Gly Thr Glu Met Ala His Ala Ala Glu Cys Ser Lys Ala Tyr Tyr Gln -5 1 5 Cys Gly Gly Lys Asn Trp Asp Gly Pro Thr Cys Cys Glu Ser Gly Ser 10 15 20 25 Thr Cys Val Asp Tyr Pro Asp Asn Pro Phe Tyr Ser Gln Cys Val Pro 30 35 40 Asn Glu Asn Leu Thr Ser Thr Asn Lys Ser Ser His Lys Thr Thr Thr 45 50 55 Thr Glu Ser Ala Lys Lys Thr Thr Thr Thr Lys Gly Ser Lys Lys Thr 60 65 70 Thr Thr Thr Glu Ala Ser Lys Lys Thr Thr Thr Thr Glu Ala Ser Lys 75 80 85 Lys Thr Thr Thr Thr Glu Ala Ser Lys Lys Thr Thr Thr Thr Thr Lys 90 95 100 105 Lys Ala Ser Thr Ser Thr Ser Ser Ser Ser Ser Ser Ala Ser Thr Asn 110 115 120 Tyr Ser Ala Val Ser Gly Gly Ala Ser Gly Asn Gly Glu Thr Thr Arg 125 130 135 Tyr Trp Asp Cys Cys Lys Pro Ser Cys Ser Trp Pro Gly Lys Ala Asp 140 145 150 Val Thr Ser Pro Val Gly Ser Cys Asn Lys Asp Gly Lys Thr Leu Ala 155 160 165 Asp Asn Asn Thr Gln Asn Gly Cys Val Gly Gly Ser Ser Tyr Thr Cys 170 175 180 185 Asn Asp Asn Gln Pro Trp Val Val Ser Asp Asp Leu Ala Tyr Gly Phe 190 195 200 Ala Ala Ala Ser Ile Ser Gly Gly Ser Glu Ala Thr Trp Cys Cys Ala 205 210 215 Cys Phe Glu Leu Thr Phe Thr Ser Thr Ala Val Lys Gly Lys Lys Met 220 225 230 Val Val Gln Val Thr Asn Thr Gly Ser Asp Leu Gly Ser Asn Thr Gly 235 240 245 Ala His Phe Asp Leu Gln Met Pro Gly Gly Gly Val Gly Ile Tyr Asn 250 255 260 265 Gly Cys Ala Thr Gln Trp Gly Ala Pro Thr Asp Gly Trp Gly Ala Arg 270 275 280 Tyr Gly Gly Val Ser Ser Ala Ser Asp Cys Ser Asn Leu Pro Ser Ala 285 290 295 Leu Gln Ala Gly Cys Lys Trp Arg Phe Gly Trp Phe Lys Asn Ala Asp 300 305 310 Asn Pro Thr Met Thr Tyr Lys Gln Val Thr Cys Pro Lys Ala Ile Thr 315 320 325 Ala Lys Ser Gly Cys Ser Arg Lys 330 335 4 338 PRT Mucor circinelloides CP99001 sig_peptide (-22)...(-1) mat_peptide (1)...(316) 4 Met Lys Phe Thr Val Ala Ile Thr Ser Ile Ala Val Ala Leu Ala Leu -20 -15 -10 Ser Ser Ser Ala Glu Ala Ala Ser Cys Ser Ser Val Tyr Gly Gln Cys -5 1 5 10 Gly Gly Ile Gly Trp Ser Gly Pro Thr Cys Cys Glu Ser Gly Ser Thr 15 20 25 Cys Val Ala Gln Glu Gly Asn Lys Tyr Tyr Ser Gln Cys Leu Pro Gly 30 35 40 Ser His Ser Asn Asn Ala Gly Asn Ala Ser Ser Thr Lys Lys Thr Ser 45 50 55 Thr Lys Thr Ser Thr Thr Thr Ala Lys Ala Thr Ala Thr Val Thr Thr 60 65 70 Lys Thr Val Thr Lys Thr Thr Thr Lys Thr Thr Thr Lys Thr Ser Thr 75 80 85 90 Thr Ala Ala Ala Ser Thr Ser Thr Ser Ser Ser Ala Gly Tyr Lys Val 95 100 105 Ile Ser Gly Gly Lys Ser Gly Ser Gly Ser Thr Thr Arg Tyr Trp Asp 110 115 120 Cys Cys Lys Ala Ser Cys Ser Trp Pro Gly Lys Ala Ser Val Thr Gly 125 130 135 Pro Val Asp Thr Cys Ala Ser Asn Gly Ile Ser Leu Leu Asp Ala Asn 140 145 150 Ala Gln Ser Gly Cys Asn Gly Gly Asn Gly Phe Met Cys Asn Asn Asn 155 160 165 170 Gln Pro Trp Ala Val Asn Asp Glu Leu Ala Tyr Gly Phe Ala Ala Ala 175 180 185 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Gly Cys Tyr Glu 190 195 200 Leu Thr Phe Thr Ser Gly Ala Ala Ser Gly Lys Lys Met Val Val Gln 205 210 215 Val Thr Asn Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Gln 220 225 230 Met Pro Gly Gly Gly Val Gly Ile Phe Asn Gly Cys Ala Ala Gln Trp 235 240 245 250 Gly Ala Pro Asn Asp Gly Trp Gly Ala Arg Tyr Gly Gly Val Ser Ser 255 260 265 Val Ser Asp Cys Ala Ser Leu Pro Ser Ala Leu Gln Ala Gly Cys Lys 270 275 280 Trp Arg Phe Asn Trp Phe Lys Asn Ser Asp Asn Pro Thr Met Thr Phe 285 290 295 Lys Glu Val Thr Cys Pro Ala Glu Leu Thr Thr Arg Ser Gly Cys Glu 300 305 310 Arg Lys 315 5 387 PRT Mucor circinelloides CP99001 sig_peptide (-22)...(-1) mat_peptide (1)...(365) 5 Met Lys Phe Thr Val Ala Ile Thr Ser Ile Ala Val Ala Leu Ala Leu -20 -15 -10 Ser Ser Ser Ala Glu Ala Ala Ser Cys Ser Ser Val Tyr Gly Gln Cys -5 1 5 10 Gly Gly Ile Gly Trp Thr Gly Pro Thr Cys Cys Asp Ala Gly Ser Thr 15 20 25 Cys Lys Ala Gln Lys Asp Asn Lys Tyr Tyr Ser Gln Cys Ile Pro Lys 30 35 40 Pro Lys Gly Ser Ser Ser Ser Ser Ser Cys Ser Ser Val Tyr Ser Gln 45 50 55 Cys Gly Gly Ile Gly Trp Ser Gly Pro Thr Cys Cys Glu Ser Gly Ser 60 65 70 Thr Cys Val Ala Gln Glu Gly Asn Lys Tyr Tyr Ser Gln Cys Leu Pro 75 80 85 90 Gly Ser His Ser Asn Asn Ala Gly Asn Ala Ser Ser Thr Lys Lys Thr 95 100 105 Ser Thr Lys Thr Ser Thr Thr Thr Ala Lys Ala Thr Ala Thr Val Thr 110 115 120 Thr Lys Thr Val Thr Lys Thr Thr Thr Lys Thr Thr Thr Lys Thr Ser 125 130 135 Thr Thr Ala Ala Ala Ser Thr Ser Thr Ser Ser Ser Ala Gly Tyr Lys 140 145 150 Val Ile Ser Gly Gly Lys Ser Gly Ser Gly Ser Thr Thr Arg Tyr Trp 155 160 165 170 Asp Cys Cys Lys Ala Ser Cys Ser Trp Pro Gly Lys Ala Ser Val Thr 175 180 185 Gly Pro Val Asp Thr Cys Ala Ser Asn Gly Ile Ser Leu Leu Asp Ala 190 195 200 Asn Ala Gln Ser Gly Cys Asn Gly Gly Asn Gly Phe Met Cys Asn Asn 205 210 215 Asn Gln Pro Trp Ala Val Asn Asp Glu Leu Ala Tyr Gly Phe Ala Ala 220 225 230 Ala Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Gly Cys Tyr 235 240 245 250 Glu Leu Thr Phe Thr Ser Gly Ala Ala Ser Gly Lys Lys Met Val Val 255 260 265 Gln Val Thr Asn Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu 270 275 280 Gln Met Pro Gly Gly Gly Val Gly Ile Phe Asn Gly Cys Ala Ala Gln 285 290 295 Trp Gly Ala Pro Asn Asp Gly Trp Gly Ala Arg Tyr Gly Gly Val Ser 300 305 310 Ser Val Ser Asp Cys Ala Ser Leu Pro Ser Ala Leu Gln Ala Gly Cys 315 320 325 330 Lys Trp Arg Phe Asn Trp Phe Lys Asn Ser Asp Asn Pro Thr Met Thr 335 340 345 Phe Lys Glu Val Thr Cys Pro Ala Glu Leu Thr Thr Arg Ser Gly Cys 350 355 360 Glu Arg Lys 365 6 346 PRT Phycomyces nitens CP99002 sig_peptide (-19)...(-1) mat_peptide (1)...(327) 6 Met Lys Phe Ser Ile Ile Ala Ser Ala Leu Leu Leu Ala Ala Ser Ser -15 -10 -5 Thr Tyr Ala Ala Glu Cys Ser Gln Gly Tyr Gly Gln Cys Gly Gly Lys 1 5 10 Met Trp Thr Gly Pro Thr Cys Cys Thr Ser Gly Phe Thr Cys Val Gly 15 20 25 Ala Glu Asn Asn Glu Trp Tyr Ser Gln Cys Ile Pro Asn Asp Gln Val 30 35 40 45 Gln Gly Asn Pro Lys Thr Thr Thr Thr Thr Thr Thr Lys Ala Ala Thr 50 55 60 Thr Thr Lys Ala Pro Val Thr Thr Thr Lys Ala Thr Thr Thr Thr Thr 65 70 75 Thr Lys Ala Pro Val Thr Thr Thr Lys Ala Thr Thr Thr Thr Thr Thr 80 85 90 Lys Thr Thr Thr Lys Thr Thr Thr Thr Lys Ala Ala Thr Thr Thr Ser 95 100 105 Ser Ser Asn Thr Gly Tyr Ser Pro Ile Ser Gly Gly Phe Ser Gly Asn 110 115 120 125 Gly Arg Thr Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys Ala Trp 130 135 140 Asp Gly Lys Ala Ser Val Thr Lys Pro Val Leu Thr Cys Ala Lys Asp 145 150 155 Gly Val Ser Arg Leu Gly Ser Asp Val Gln Ser Gly Cys Val Gly Gly 160 165 170 Gln Ala Tyr Met Cys Asn Asp Asn Gln Pro Trp Val Val Asn Asp Asp 175 180 185 Leu Ala Tyr Gly Phe Ala Ala Ala Ser Leu Gly Ser Ala Gly Ala Ser 190 195 200 205 Ala Phe Cys Cys Gly Cys Tyr Glu Leu Thr Phe Thr Asn Thr Ala Val 210 215 220 Ala Gly Lys Lys Phe Val Val Gln Val Thr Asn Thr Gly Asp Asp Leu 225 230 235 Ser Thr Asn His Phe Asp Leu Gln Met Pro Gly Gly Gly Val Gly Tyr 240 245 250 Phe Asn Gly Cys Gln Ser Gln Trp Asn Thr Asn Thr Asp Gly Trp Gly 255 260 265 Ala Arg Tyr Gly Gly Ile Ser Ser Ile Ser Glu Cys Asp Lys Leu Pro 270 275 280 285 Thr Gln Leu Gln Ala Gly Cys Lys Trp Arg Phe Gly Trp Phe Lys Asn 290 295 300 Ala Asp Asn Pro Glu Val Thr Phe Lys Ala Val Thr Cys Pro Ala Glu 305 310 315 Ile Ile Ala Lys Thr Gly Cys Glu Arg Lys 320 325 7 1257 DNA Humicola insolens intron (453)..(509) 7 aatgacgggg caacctcccg cccgggccca actcttgggt ttggtttgac aggccgtctg 60 tctcttgcgt cctcttacta cgcctgcctg gaccctacgt ctcaactccg attcaagatg 120 cgttcctccc ctctcctccg ctccgccgtt gtggccgccc tgccggtgtt ggcccttgcc 180 gctgatggca agtccacccg ctactgggac tgctgcaagc cttcgtgcgg ctgggccaag 240 aaggctcccg tgaaccagcc tgtcttctcc tgcaacgcca acttccagcg tctcactgac 300 ttcgacgcca agtccggctg cgagccgggc ggtgtcgcct actcgtgcgc cgaccagacc 360 ccatgggctg tgaacgacga cttcgcgttc ggttttgctg ccacctctat tgccggcagc 420 aatgaggcgg gctggtgctg cgcctgctac gagtaagctt tggtcgcgtg tgtaacactg 480 tgcaggcata gcactaacca cctcccaggc tcaccttcac atccggtcct gttgctggca 540 agaagatggt cgtccagtcc accagcactg gcggtgatct tggcagcaac cacttcgatc 600 tcaacatccc cggcggcggc gtcggcatct tcgacggatg cactccccag ttcggcggtc 660 tgcccggcca gcgctacggc ggcatctcgt cccgcaacga gtgcgatcgg ttccccgacg 720 ccctcaagcc cggctgctac tggcgcttcg actggttcaa gaacgccgac aacccgagct 780 tcagcttccg tcaggtccaa tgcccagccg agctcgtcgc tcgcaccgga tgccgccgca 840 acgacgacgg caacttccct gccgtccaga tcccctccag cagcaccagc tctccggtcg 900 gccagcctac cagtaccagc accacctcca cctccaccac ctcgagcccg cccgtccagc 960 ctacgactcc cagcggctgc actgctgaga ggtgggctca gtgcggcggc aatggctgga 1020 gcggctgcac cacctgcgtc gctggcagca cctgcacgaa gattaatgac tggtaccatc 1080 agtgcctgta aacgcagggc agcctgagaa ccttactggt tgcgcaacga aatgacactc 1140 ccaatcactg tattagttct tgtacataat ttcgtcatcc ctccagggat tgtcacatat 1200 atgcaatgat gaatactgaa cacaaacctg gccgcttgaa ctggccgaag gaatgcc 1257 8 1720 DNA Trichoderma viride intron (500)..(682) 8 ggtgtgtcat ttctcctcaa catactgcct ttcaacaact ttcgcctcct ccctggcctg 60 atatcccaat atcagttttt cccaaagtag caagtcatca gtaaatctgc tcatctatca 120 ttaatcagtg cccatagtgt ctgtctgttg attgcctccc gccatacacg atgaacagga 180 ccatggctcc attgctgctt gcagcgtcga tactcttcgg gggcgctgct gcacaacaga 240 ctgtctgggg acagtgtgga ggtattggtt ggagcggacc tacgagttgt gctcctggat 300 cagcttgttc tactctcaat ccttattatg cgcaatgcat tccgggggcc actagtatca 360 ccacctcgac ccgacccccc tcgggtccaa ccaccaccac cagagccacc tcaacgacct 420 catctccgcc accgaccagc tctggagttc gatttgctgg cgttaacatc gcgggctttg 480 acttcggatg taccacagag tatgtcttca tgttgcatag tgttgctggc tgagtattct 540 gggcggatga tttatagctg tgcgggctgc aaaacaccgc cggtctgcca ctatcaaggc 600 atagttgata ggcggcggtg ttttcttcaa tcccctgatt acactctcaa gaatctagtg 660 gctgatggat gtatgattac agtggcactt gcgttacatc gaaggtttat cctccgttga 720 agaacttcac tggggcaaac aactacccgg acggtatcgg ccagatgcag cacttcgtca 780 acgatgatgg gatgactatt ttccgcctac ccgtcggatg gcagtacctc gtaaacaaca 840 atctgggtgg aactctcgat tccaccagta tctcgaagta tgatcagctc gttcaggggt 900 gcctgtctct cggtgtatac tgcatcatcg acatccacaa ttatgctcga tggaacggtg 960 gaatcattgg ccagggaggc cctacaaatg cccagtttac cagtctttgg tcgcagttgg 1020 catcgaagta cgcgtctcag tcgagggtgt ggttcggaat aatgaatgag ccccacgacg 1080 tgaacatcaa cacttgggct gccacggttc aagaggtcgt cactgcaatc cgcaacgccg 1140 gtgctacgtc gcaatacatt tctctgcctg gaaatgatta tcaatctgcg gcagctttta 1200 tttccgatgg cagtgcagcc gccctgtctc aggtaacgaa ccctgatgga tcaacaacga 1260 atctaatctt cgatgtccac aagtacttag actcggacaa ctccggtact cacgccgaat 1320 gcactacaaa caacatcgac ggcgcctttg ctcctctcgc cacttggctt cgacagaaca 1380 accgccaggc tattctgacg gaaaccggcg gtggcaatgt tcagtcctgc atccaagatt 1440 tgtgccaaca gatccagtac ctcaaccaga actcagatgt ctatcttggc tatgctggct 1500 ggggtgccgg ttcatttgat agcacttata ttctgacgga aacgcctact ggaagcggta 1560 actcgtggac ggacacatcc ctagttagct cgtgtctcgc caggaagtaa caccgaggtc 1620 gattgcagga gccttgtcaa tagcgatttc atcttgctgt acataattct tactctctga 1680 agccgcttgt tctgggtatg tgtcttgaca ggtttctaga 1720 

1. A cellulase preparation comprising nonionic surfactant(s) together with endoglucanase(s) derived from Zygomycetes.
 2. The cellulase preparation according to claim 1 wherein the Zygomycetes is a microorganism belonging to Rhizopus sp., Mucor sp., or Phycomyces sp.
 3. A cellulase preparation comprising nonionic surfactant(s) together with protein(s) (a) and/or (b) as described below: (a) a protein comprising an amino acid sequence as shown in any one of SEQ ID NOS: 1 to 6, and (b) a protein comprising an amino acid sequence as shown in any one of SEQ ID NOS: 1 to 6, having addition, insertion, deletion or substitution of one or more amino acids, and having endoglucanase activity.
 4. A cellulase preparation comprising nonionic surfactant(s) together with protein(s) encoded by a gene comprising DNA (a) or (b) as described below: (a) a DNA encoding an amino acid sequence as shown in any one of SEQ ID NOS: 1 to 6, and (b) a DNA complementary to a DNA hybridizing to a DNA encoding a protein comprising an amino acid sequence as shown in any one of SEQ ID NOS: 1 to 6 under stringent conditions, and which encodes a protein having endoglucanase activity.
 5. The cellulase preparation according to any one of claims 1 to 4 wherein the nonionic surfactant is polyoxyethylene alkylether, polyoxyethylene alkylphenylether, polyoxyethylene monofatty acid ester, polyoxyethylene sorbitan monofatty acid ester, sorbitan monofatty acid ester, polyethylene glycol, glycerol monofatty acid ester, polyglycerine fatty acid ester, alkyl glycoside, esters of polyethoxylated alkyl glycosides, alkyl dimethyl amine oxide, fatty acid diethanol amide, polyoxyethylene alkylamine, terephthalic acid tetraethylene glycol polymer, alkyl polyethylene glycol ether, nonyl phenol polyethylene glycol ether, or fatty acid ester of sucrose or glucose.
 6. The cellulase preparation according to any one of claims 1 to 5 comprising 0.1 to 50% by weight of nonionic surfactant(s).
 7. The cellulase preparation according to any one of claims 1 to 6, which is a non-dusting-granule or stabilized liquid.
 8. A detergent composition comprising the cellulase preparation according to any one of claims 1 to 7 with detergent components.
 9. A method of treating fabric which comprises treating cellulose-containing fabric with the cellulase preparation according to any one of claims 1 to 7 so as to improve the properties of the fabric.
 10. The method according to claim 9 wherein the improvement of the properties of the fabric is the color clarification.
 11. The method according to claim 9 wherein the improvement of the properties of the fabric is the removal of fuzz.
 12. The method according to claim 9 wherein the improvement of the properties of the fabric is the providing of stonewash-like appearance and texture.
 13. The method according to claim 9 wherein the improvement of the properties of the fabric is the improvement of touch and appearance.
 14. The method according to claim 9 wherein the improvement of the properties of the fabric is softening of the fabric.
 15. The method according to claim 9 wherein nonionic surfactant(s) are present in a concentration of 10 to 10,000 mg/L in a reaction system.
 16. The method according to claim 9 wherein the method is carried out through a step of soaking, washing or rinsing the fabric.
 17. A method of deinking waste paper comprising a step of treating waste paper with the cellulase preparation according to any one of claims 1 to 7 together with a deinking agent.
 18. A method of improving the freeness of paper pulp comprising a step of treating paper pulp with the cellulase preparation according to any one of claims 1 to
 7. 